QUIDEL MOLECULAR RSV + HMPV ASSAY

K122189 · Quidel Corp. · OEM · Mar 8, 2013 · Microbiology

Device Facts

• Record ID: K122189
• Device Name: QUIDEL MOLECULAR RSV + HMPV ASSAY
• Applicant: Quidel Corp.
• Product Code: OEM · Microbiology
• Decision Date: Mar 8, 2013
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3980
• Device Class: Class 2

Indications for Use

The Quidel Molecular RSV + hMPV assay is a multiplex Real Time RT-PCR assay for the qualitative detection and identification of respiratory syncytial virus and human metapneumovirus RNA extracted from nasal and nasopharyngeal swabs specimens from patients with signs and symptoms of respiratory infection. This in vitro diagnostic test is intended to aid in the differential diagnosis of respiratory syncytial virus and human metapneumovirus infections in humans in conjunction with clinical and epidemiological risk factors. This test is not intended to differentiate the two subtypes of RSV or the four genetic sub-lineages of hMPV. Negative results do not preclude RSV infection and/or hMPV infection and should not be used as the sole basis for diagnosis, treatment or other patient management decisions.

Device Story

The Quidel Molecular RSV + hMPV Assay is an in vitro diagnostic test for the qualitative detection of RSV and hMPV viral RNA. Input consists of nasal or nasopharyngeal swab specimens. Nucleic acids are extracted using the automated NucliSENS easyMAG platform. The assay utilizes a multiplex Real-Time RT-PCR reaction performed on either the Cepheid SmartCycler II or the Applied Biosystems 7500 Fast Dx instrument. The process involves rehydrating a lyophilized Master Mix containing target-specific primers and fluorescent-labeled probes. The system uses TaqMan chemistry with an enzyme possessing reverse transcriptase, DNA polymerase, and 5'-3' exonuclease activities. During amplification, probes are cleaved, releasing reporter dyes and generating a fluorescent signal. The device is used in clinical laboratory settings by trained personnel. Output is the detection or absence of viral RNA, which aids clinicians in the differential diagnosis of respiratory infections. It does not differentiate RSV subtypes or hMPV sub-lineages. Results are intended to be used in conjunction with other clinical and epidemiological findings to inform patient management decisions.

Clinical Evidence

Prospective clinical study (2012 season, n=1014). Compared against DSFA/culture (RSV) and FDA-cleared molecular test (hMPV). RSV sensitivity: 97.9-98.6%; specificity: 96.8-97.6%. hMPV positive percent agreement: 96.7-98.0%; negative percent agreement: 98.9-99.6%. Discordant RSV samples confirmed positive by secondary FDA-cleared RT-PCR.

Technological Characteristics

Nucleic acid-based RT-PCR; multiplex assay using sequence-specific fluorescent-labeled probes. Instrumentation: Cepheid SmartCycler II or ABI 7500 Fast Dx. Extraction: bioMérieux NucliSENS easyMAG. Targets: RSV (L viral polymerase and NS2 genes) and hMPV (RNA polymerase gene).

Indications for Use

Indicated for patients with signs and symptoms of respiratory infection. Used for qualitative detection and identification of RSV and hMPV viral RNA from nasal and nasopharyngeal swab specimens to aid in differential diagnosis.

Regulatory Classification

Identification

A respiratory viral panel multiplex nucleic acid assay is a qualitative in vitro diagnostic device intended to simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or viral culture. The detection and identification of a specific viral nucleic acid from individuals exhibiting signs and symptoms of respiratory infection aids in the diagnosis of respiratory viral infection when used in conjunction with other clinical and laboratory findings. The device is intended for detection and identification of a combination of the following viruses:(1) Influenza A and Influenza B; (2) Influenza A subtype H1 and Influenza A subtype H3; (3) Respiratory Syncytial Virus subtype A and Respiratory Syncytial Virus subtype B; (4) Parainfluenza 1, Parainfluenza 2, and Parainfluenza 3 virus; (5) Human Metapneumovirus; (6) Rhinovirus; and (7) Adenovirus.

Special Controls

*Classification.* Class II (special controls). The special controls are:(1) FDA's guidance document entitled “Class II Special Controls Guidance Document: Respiratory Viral Panel Multiplex Nucleic Acid Assay;” (2) For a device that detects and identifies Human Metapneumovirus, FDA's guidance document entitled “Class II Special Controls Guidance Document: Testing for Human Metapneumovirus (hMPV) Using Nucleic Acid Assays;” and (3) For a device that detects and differentiates Influenza A subtype H1 and subtype H3, FDA's guidance document entitled “Class II Special Controls Guidance Document: Testing for Detection and Differentiation of Influenza A Virus Subtypes Using Multiplex Nucleic Acid Assays.” See § 866.1(e) for the availability of these guidance documents.

Predicate Devices

• ProFlu™ Assay (k092500)
• Pro hMPV+ Assay (k082688)

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

A. 510(k) Number: K122189

B. Purpose for Submission: The purpose of this submission is to show that the Quidel Molecular RSV + hMPV Assay is substantially equivalent to Prodesse’s ProFlu™ Assay and Pro hMPV+ Assay. The ProFlu Assay detects RSV RNA while the Pro hMPV+ assay detects hMPV RNA.

C. Measurand: This assay detects the presence of RSV and hMPV nucleic acid using RT-PCR. Both viruses have negative single-stranded RNA genomes. The RT-PCR primers are developed to bind to conserved regions of L viral polymerase and NS2 genes for RSV and RNA polymerase gene for hMPV.

D. Type of Test: This is a nucleic acid based test using reverse transcription polymerase chain reaction (RT-PCR). The negative sense RNA genomes of RSV and hMPV are reverse transcribed and then amplified during the RT-PCR reaction. The presence of RSV or hMPV RNA is then detected through sequence-specific labeled probe that is cleaved during PCR amplification releasing the fluorescence reporter dye from the quencher dye.

E. Applicant: Quidel Corporation

F. Proprietary and Established Names: Quidel® Molecular RSV + hMPV Assay

G. Regulatory Information:

1. Regulation section: 21 CFR866.3980, Respiratory viral panel multiplex nucleic acid assay
2. Classification: Class II
3. Product code: OEM, OCC
4. Panel: Microbiology (83)

H. Intended Use:

1. Intended use(s):

The Quidel Molecular RSV + hMPV assay is a multiplex Real Time RT-PCR assay for the qualitative detection and identification of respiratory syncytial virus and human metapneumovirus RNA extracted from nasal and nasopharyngeal swabs specimens from patients with signs and symptoms of respiratory infection. This in vitro diagnostic test is intended to aid in the differential diagnosis of respiratory syncytial virus and human metapneumovirus infections in humans in conjunction with clinical and epidemiological risk factors. This test is not intended to differentiate the two subtypes of RSV or the four genetic sub-lineages of hMPV.

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Negative results do not preclude RSV infection and/or hMPV infection and should not be used as the sole basis for diagnosis, treatment or other patient management decisions.

2. Indication(s) for use:
Same as intended use.

3. Special conditions for use statement(s):
Prescription only.

4. Special instrument requirements:
Cepheid SmartCycler II or ABI 7500 Fast Dx

I. Device Description: The Quidel Molecular RSV + hMPV Assay detects viral nucleic acids that have been extracted from a patient sample using the NucliSENS® easyMAG® automated extraction platform. An RT-PCR reaction is then performed in a single tube generating amplicons for each of the target viruses present in the sample. This reaction is performed utilizing either the Cepheid SmartCycler® II or the Applied Biosystems 7500 Fast DX. Identification of RSV and hMPV and the PRC occurs by the use of target-specific primers and fluorescent-labeled probes that hybridize to conserved regions in the genomes of RSV and hMPV and the PRC. Results are analyzed by either the Cepheid or ABI software and reported to the user.

J. Substantial Equivalence Information:

1. Predicate device name(s):
ProFlu™ Assay, Pro hMPV+ Assay

2. Predicate 510(k) number(s):
k092500, k082688

3. Comparison with predicate:

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| Subject Device and Comparator Device Comparison | | | |
| --- | --- | --- | --- |
| Item | Subject Device
Quidel Molecular RSV + hMPV Assay | Predicate Device
Prodesse ProFlu+ | Predicate Device
Prodesse ProhMPV+ |
| Intended Use | The Quidel Molecular RSV + hMPV assay is a multiplex Real Time RT-PCR assay for the in vitro qualitative detection and identification of respiratory syncytial virus and human metapneumovirus viral RNA extracted from nasal and nasopharyngeal swabs specimens with signs and symptoms of respiratory infection. This in vitro diagnostic test is intended to aid in the differential diagnosis of respiratory syncytial virus and human metapneumovirus infections. This test is not intended to differentiate the four genetic sub-lineages of hMPV.

Negative results do not preclude RSV or hMPV infection and should not be used as the sole basis for diagnosis, treatment or other patient management decisions. | The ProFlu™+ Assay is a multiplex Real-Time PCR (RT-PCR) in vitro diagnostic test for the rapid and qualitative detection and discrimination of Influenza A Virus, Influenza B Virus, and Respiratory Syncytial Virus (RSV) nucleic acids isolated and purified from nasopharyngeal (NP) swab specimens obtained from symptomatic patients. This test is intended for use to aid in the differential diagnosis of Influenza A, Influenza B and RSV viral infections in humans and is not intended to detect Influenza C.

Negative results do not preclude influenza or RSV virus infection and should not be used as the sole basis for treatment or other management decisions. It is recommended that negative RSV results be confirmed by culture.

Performance characteristics for Influenza A Virus were established when Influenza A/H3 and | The Pro hMPV+ Assay is a Real Time RT-PCR in vitro diagnostic test for the qualitative detection of human Metapneumovirus (hMPV) nucleic acid isolated and purified from nasopharyngeal swab (NP) specimens obtained from individuals exhibiting signs and symptoms of acute respiratory infection. This assay targets a highly conserved region of the Nucleocapsid gene of hMPV. The detection of hMPV nucleic acid from symptomatic patients aids in the diagnosis of human respiratory hMPV infection if used in conjunction with other clinical and laboratory findings. This test is not intended to differentiate the four genetic sub-lineages of hMPV.

Negative results do not preclude hMPV infection and should not be used as the sole basis for diagnosis, treatment or other management decisions. |

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| Subject Device and Comparator Device Comparison | | | |
| --- | --- | --- | --- |
| Item | Subject Device
Quidel Molecular RSV
+ hMPV Assay | Predicate Device
Prodesse ProFlu+ | Predicate Device
Prodesse ProhMPV+ |
| | | A/H1 were the predominant Influenza A viruses in circulation. When other Influenza A viruses are emerging, performance characteristics may vary.

If infection with a novel Influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent Influenza viruses and sent to state or local health department for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens. | |
| Assay Target | Respiratory syncytial virus, hMPV | Influenza A virus, influenza B virus, respiratory syncytial virus | hMPV |
| Sample Types | Nasal swab, nasopharyngeal swab | Nasopharyngeal swab | Nasopharyngeal swab |
| Extraction Methods | bioMérieux easyMAG
Automated Magnetic Extraction Reagents | Roche MagNA Pure LC
Total Nucleic Acid Isolation Kit or the bioMérieux easyMAG | Roche MagNA Pure LC
Total Nucleic Acid Isolation Kit or the bioMérieux easyMAG |

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| Subject Device and Comparator Device Comparison | | | |
| --- | --- | --- | --- |
| Item | Subject Device
Quidel Molecular RSV
+ hMPV Assay | Predicate Device
Prodesse ProFlu+ | Predicate Device
Prodesse ProhMPV+ |
| | | Automated Magnetic Extraction Reagents | Automated Magnetic Extraction Reagents |
| Assay Methodology | PCR-based system for detecting the presence or absence of viral RNA in clinical specimens | PCR-based system for detecting the presence or absence of viral RNA in clinical specimens | PCR-based system for detecting the presence or absence of viral RNA in clinical specimens |
| Detection Techniques | Multiplex assay using different reporter dyes for each target | Multiplex assay using different reporter dyes for each target | Multiplex assay using different reporter dyes for each target |
| Viral Targets | RSV: L viral polymerase and NS2 genes
hMPV: RNA polymerase gene | Influenza A: Matrix gene; Influenza B: Non-structural NS1 and NS2 | Nucleocapsid |
| LoD | The analytical sensitivity (limit of detection or LoD) of the Quidel Molecular hMPV assay was determined using quantified (TCID_{50}/mL) cultures of 2 RSV strains and 4 hMPV strains (A1, A2, B1, B2) serially diluted in negative nasopharyngeal matrix. Each dilution was extracted using the NucliSENS easyMAG System and tested in replicates of 20 per concentration of virus on both the Applied Biosystems® 7500 Fast Dx platform. Analytical sensitivity (LoD) is defined as the lowest concentration at which 95% of all replicates | The analytical sensitivity (limit of detection or LoD) of the ProFlu+ Assay was determined using quantified (TCID_{50}/mL) cultures of 4 Influenza A (2 H1N1 and 2 H3N2), 2 Influenza B, 2 Respiratory Syncytial Virus Type A, and 2 Respiratory Syncytial Virus Type B strains serially diluted in nasopharyngeal clinical matrix. Each viral strain was extracted using the Roche MagNA Pure LC instrument and tested in replicates of 20 per concentration of virus. Analytical sensitivity (LoD), as defined as the lowest concentration at which ≥ 95% of all replicates tested positive, ranged from 10^{2} – 10^{1} TCID_{50}/mL. | The analytical sensitivity (limit of detection or LoD) of the Pro hMPV+ Assay was determined using quantified (TCID_{50}/mL) cultures of 2 hMPV (subtype A2 and subtype B2) strains serially diluted in nasopharyngeal clinical matrix. Each viral strain was extracted using the Roche MagNA Pure LC instrument and tested in replicates of 20 per concentration of virus. Analytical sensitivity (LoD) as defined as the lowest concentration at which ≥ 95% of all replicates tested positive, ranged from 10^{2} – 10^{1} TCID_{50}/mL. |

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| Subject Device and Comparator Device Comparison | | | |
| --- | --- | --- | --- |
| Item | Subject Device
Quidel Molecular RSV
+ hMPV Assay | Predicate Device
Prodesse ProFlu+ | Predicate Device
Prodesse ProhMPV+ |
| | tested positive, ranged from 10^{-1} to 10^{1} TCID_{50}/mL. | ranged from 10^{2} to 10^{-1} TCID_{50}/mL. | |

K. Standard/Guidance Document Referenced (if applicable): N/A

L. Test Principle:
This is a nucleic acid based test using reverse transcription polymerase chain reaction (RT-PCR). Viral infection is detected through the use of PCR to amplify and detect viral RNA. The negative sense RNA genomes of RSV and hMPV are reverse-transcribed and then amplified during the RT-PCR reaction. The presence of RSV or hMPV RNA is then detected through sequence-specific labeled probe that is cleaved during PCR amplification releasing the fluorescence reporter dye from the quencher dye.

M. Performance Characteristics (if/when applicable):

1. Analytical performance:

a. Precision/Reproducibility:
Reproducibility studies were performed on both thermal cycler instruments, the Cepheid SmartCycler II and ABI 7500 Fast Dx. The studies were conducted at three separate testing sites where two users tested each virus sample in triplicate over 5 days. The hMPV-A2 or RSV A viruses were tested at High Negative (0.05xLoD), Low Positive, close to the detection limit (2x LoD) and Medium Positive (5x LoD) concentrations. The viruses were diluted in a negative nasal matrix. Negative and positive controls were included in the study. The positive control was each virus diluted in UTM and the negative controls were UTM only (Negative control) and negative nasal matrix (RSV and hMPV negative). To conduct each test, the contrived samples were extracted using the bioMérieux easyMAG system and tested on either the Cepheid SmartCycler II or the ABI 7500 Fast Dx.

| Reproducibility Results – Cepheid SmartCycler II | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Panel Member ID | Site 1 | | | Site 2 | | | Site 3 | | | Total Positive Results |
| | Positive Results | AVE Ct* | %CV | Positive Results | AVE Ct* | %CV | Positive Results | AVE Ct* | %CV | |
| RSV High Negative 0.05x LoD | 8/30 | 44.3 | 9.3 | 6/30 | 47.4 | 5.4 | 1/30 | 48.7 | N/A | 15/90 |

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| Reproducibility Results – Cepheid SmartCycler II | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Panel Member ID | Site 1 | | | Site 2 | | | Site 3 | | | Total Positive Results |
| | Positive Results | AVE Ct* | %CV | Positive Results | AVE Ct* | %CV | Positive Results | AVE Ct* | %CV | |
| RSV Low Positive 2x LoD | 30/30 | 31.2 | 7.3 | 30/30 | 31.4 | 4.1 | 30/30 | 30.9 | 1.5 | 90/90 |
| RSV Med Positive 5x LoD | 30/30 | 29.6 | 5.7 | 30/30 | 29.7 | 3.1 | 30/30 | 29.5 | 2.0 | 90/90 |
| RSV Negative | 0/30 | N/A | N/A | 0/30 | N/A | N/A | 0/30 | N/A | N/A | 0/90 |
| RSV Negative Control | 0/30 | N/A | N/A | 0/30 | N/A | N/A | 0/30 | N/A | N/A | 0/90 |
| RSV Positive Control | 30/30 | 30.0 | 1.8 | 30/30 | 31.0 | 3.3 | 30/30 | 30.6 | 1.2 | 90/90 |
| hMPV High Negative 0.15x LoD | 12/30 | 34.3 | 8.3 | 6/30 | 41.8 | 6.3 | 8/30 | 34.9 | 6.4 | 26/90 |
| hMPV Low Positive 2x LoD | 30/30 | 31.1 | 6.7 | 30/30 | 32.2 | 6.2 | 30/30 | 31.1 | 3.3 | 90/90 |
| hMPV Med Positive 5x LoD | 30/30 | 29.3 | 2.0 | 30/30 | 29.9 | 3.2 | 30/30 | 29.8 | 3.4 | 90/90 |
| hMPV Negative | 0/30 | N/A | N/A | 0/30 | N/A | N/A | 0/30 | N/A | N/A | 0/90 |
| hMPV Negative Control | 0/30 | N/A | N/A | 0/30 | N/A | N/A | 0/30 | N/A | N/A | 0/90 |
| hMPV Positive Control | 30/30 | 30.3 | 1.4 | 30/30 | 30.9 | 1.9 | 30/30 | 30.8 | 2.5 | 90/90 |
| Reproducibility Results – Applied Biosystems 7500 Fast Dx | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Panel Member ID | Site 1 | | | Site 2 | | | Site 3 | | | Total Positive Results |
| | Positive Results | AVE Ct* | %CV | Positive Results | AVE Ct* | %CV | Positive Results | AVE Ct* | %CV | |
| RSV High Negative 0.15x LoD | 14/30 | 30.7 | 8.3 | 3/30 | 33.8 | 2.4 | 9/30 | 32.3 | 4.0 | 26/90 |
| RSV Low Positive | 30/30 | 23.7 | 7.4 | 30/30 | 23.8 | 3.1 | 30/30 | 23.4 | 4.5 | 90/90 |

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| Reproducibility Results – Applied Biosystems 7500 Fast Dx | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Panel | Site 1 | | | Site 2 | | | Site 3 | | | Total |
| 2x LoD | | | | | | | | | | |
| RSV Med Positive 5x LoD | 30/30 | 20.5 | 4.0 | 30/30 | 21.8 | 2.2 | 29/29 | 21.1 | 2.2 | 89/89 |
| RSV Negative | 0/30 | N/A | N/A | 0/30 | N/A | N/A | 0/30 | N/A | N/A | 0/90 |
| RSV Negative Control | 0/30 | N/A | N/A | 0/30 | N/A | N/A | 0/30 | N/A | N/A | 0/90 |
| RSV Positive Control | 30/30 | 19.7 | 6.5 | 30/30 | 20.0 | 8.5 | 30/30 | 19.6 | 1.9 | 90/90 |
| hMPV High Negative 0.15x LoD | 14/30 | 24.9 | 19.5 | 6/30 | 29.1 | 10.2 | 10/30 | 25.1 | 16.9 | 30/90 |
| hMPV Low Positive 2x LoD | 30/30 | 21.0 | 8.7 | 30/30 | 21.5 | 4.1 | 30/30 | 21.6 | 5.8 | 90/90 |
| hMPV Med Positive 5x LoD | 30/30 | 18.9 | 3.3 | 30/30 | 19.6 | 3.0 | 29/29 | 19.2 | 2.6 | 89/89 |
| hMPV Negative | 0/30 | N/A | N/A | 0/30 | N/A | N/A | 0/30 | N/A | N/A | 0/90 |
| hMPV Negative Control | 0/30 | N/A | N/A | 0/30 | N/A | N/A | 0/30 | N/A | N/A | 0/90 |
| hMPV Positive Control | 30/30 | 19.7 | 3.9 | 30/30 | 20.0 | 1.7 | 30/30 | 19.9 | 1.4 | 90/90 |

- Average Ct based on the average of only positive results. Ct values of 0 were left out of this calculation.

The data reported shows 100% detection of hMPV and RSV at Low and Medium Positive levels demonstrating good reproducibility of the test. Detection was lower than 50% for the High Negative samples which is acceptable and typical for nucleic acid tests.

b. Linearity/assay reportable range: N/A

c. Traceability, Stability, Expected values (controls, calibrators, or methods): A Fresh vs. Frozen study was performed using the same contrived specimens in negative clinical

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matrix as was used in the Reproducibility Study. Briefly, hMPV-A2 and RSV were diluted into negative clinical matrix at 5x LoD, 2x LoD and 0.15x LoD. The viral panel was tested before freezing and then after frozen storage at -70°C for 22-56 days; each sample was tested in triplicate. No intermediate time points were tested for the stability studies. Additionally, 99 clinical samples were tested within 72 hours of collection and once again after 7 days at -70°C.

| | Applied Biosystems 7500 Fast Dx Data | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | RSV | | | | hMPV | | | |
| | | 5x LoD | 2x LoD | 0.15x LoD | Negative | 5x LoD | 2x LoD | 0.15x LoD | Negative |
| Fresh Testing | Average | 20.9 | 22.7 | 29.6 | N/A | 18.4 | 19.2 | 25.0 | N/A |
| | STDEV | 0.5 | 0.6 | 2.0 | N/A | 0.5 | 0.3 | 2.8 | N/A |
| | %CV | 2.6% | 2.8% | 6.8% | N/A | 2.6% | 1.6% | 11.1% | N/A |
| Post-Freeze | Average | 21.7 | 23.0 | 32.4 | N/A | 19.5 | 21.0 | 27.7 | N/A |
| | STDEV | 0.3 | 0.4 | 1.9 | N/A | 0.2 | 0.7 | 3.4 | N/A |
| | %CV | 1.3% | 1.7% | 5.9% | N/A | 1.1% | 3.1% | 12.4% | N/A |

Sample storage at -70°C for up to 56 days did not impact the ability of the Quidel Molecular RSV + hMPV Assay to detect either RSV or hMPV positive near the clinical cut-off. 2x and 5x LoD samples were both detected 100% (90/90) of the time. There was also no statistical difference in Ct value before and after freezing for the 2x LoD and 5x LoD samples. The data of the 99 clinical samples, not shown here, also did not show any significant change in Ct value over time when samples were stored at -70°C.

Stability studies were also performed on rehydrated master mix and extracted RNA samples. hMPV or RSV viruses at 3x LoD were added to negative sample matrix, extracted for RNA and the RT-PCR reaction was performed; the master mix study was performed in triplicate while the RNA extraction study was performed in quadruplicate. The master mix was stable at room temperature and 2° to 8°C for 2 days and for 5 days at -20°C. Extracted RNA stability studies showed that hMPV and RSV extracted RNA was stable for 6 hours at 2° to 8°C, 4 hours at room temperature, one month at -20°C and for 96 hours at -20°C with up to 3 freeze/thaw cycles.

There was no significant change in Ct value during any of the stability studies which supports the stability claims.

d. Detection limit:

The LoD was determined for hMPV and RSV using each of the 4 genetic sub-lineages and 2 subtypes of each virus respectively. Virus was diluted in pooled negative NP swab clinical matrix. Each virus sub-lineage/subtype was diluted and each dilution was extracted and run 20 times using both the Cepheid SmartCycler II and ABI 7500 Fast Dx, The LoD was determined to be the lowest titer at which the virus was detected greater than 95% of the time (19/20 or 20/20). For hMPV-A2, hMPV-B2, RSV A and RSV B three separate kit lots were tested and a representative titer was chosen that showed the best repeatability. The final LoD titers were reported for each instrument and all future calculations used the LoD for the specific virus and thermal cycler on which an experiment was being conducted.

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| Virus | ABI 7500 Fast Dx | | | Cepheid SmartCycler II | | |
| --- | --- | --- | --- | --- | --- | --- |
| | Lot 1 (110411) | Lot 2 (2N0020) | Lot 3 (908842) | Lot 1 (110411) | Lot 2 (2N0020) | Lot 3 (908842) |
| | LoD TCID_{50}/mL | LoD TCID_{50}/mL | LoD TCID_{50}/mL | LoD TCID_{50}/mL | LoD TCID_{50}/mL | LoD TCID_{50}/mL |
| hMPV-A1 | 1.76E+01 | | | 2.65E+01 | | |
| hMPV-A2 | 4.17E+00 | 4.17E+00 | 1.25E+00 | 4.17E+00 | 4.17E+00 | 4.17E+00 |
| hMPV-B1 | 1.05E+00 | | | 7.88E+00 | | |
| hMPV-B2 | 3.21E+00 | 3.21E+00 | 3.21E+00 | 3.21E+00 | 9.63E+00 | 3.21E+00 |
| | | | | | | |
| RSV A | 6.29E-01 | 5.10E-01 | 5.10E-01 | 1.89E+00 | 1.70E+00 | 5.10E-01 |
| RSV B | 7.5E-01 | 7.5E-01 | 7.5E-01 | 7.5E-01 | 2.25E+00 | 2.25E+00 |

The reported LoD values for each instrument are as follows:

| LoD Value Summary for the 7500 Fast Dx & SmartCycler | | |
| --- | --- | --- |
| Virus | LoD TCID50/mL 7500 Fast Dx | LoD TCID50/mL SmartCycler |
| RSV A | 6.29E-01 | 1.89E+00 |
| RSV B | 7.50E-01 | 2.25E+00 |
| | | |
| hMPV-A1 | 1.76E+01 | 2.65E+01 |
| hMPV-A2 | 4.17E+00 | 4.17E+00 |
| hMPV-B1 | 1.05E+00 | 7.88E+00 |
| hMPV-B2 | 3.21E+00 | 9.63E+00 |

e. Analytical specificity:

Interference:

To evaluate the potential interference of other common respiratory pathogens the organisms in the table below were tested. Samples were prepared by adding RSV B or hMPV-B2 and the potential interference organism into negative clinical matrix at 3x LoD. The potential interference organisms were tested at one clinically relevant concentration, for viruses $10^{4}-10^{6}$ TCID50/mL and for bacteria $10^{6}-10^{8}$ CFU/mL. Additionally RSV and hMPV were tested for interference when both organisms are in the same sample. For the intra test interference RSV B was tested at 33,300x LoD, 333x LoD, 100x LoD, 30x LoD and 3x LoD. hMPV-B2 was tested at 10,000x LoD, 100x LoD, 30x LoD, 3x LoD. The prepared samples were extracted and run on both the Cepheid SmartCycler II and ABI 7500 Fast Dx.

There was no interference seen between any of the organisms tested, all tests were positive for either RSV or hMPV. A decrease in Ct value was seen in the presence of Neisseria gonorrhoeae, but sequence analysis showed no homology between the test probe sequence and Neisseria gonorrhoeae sequence. At very high levels (at or above 1,000x LoD) RSV may interfere with the detection of hMPV. However, during the clinical trial co-infection was found

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to be a rare event, and the concentrations used when interference occurred are much higher than typically present in clinical specimens. Therefore, the risk of RSV/hMPV interference is very low.

| Organism | Final Concentration | RSV detection | hMPV detection |
| --- | --- | --- | --- |
| hMPV A2 | 3xLoD | - | + |
| hMPV B2 | 3xLoD | - | + |
| RSV A Long | 3xLoD | + | - |
| RSV B Wash | 3xLoD | + | - |
| A/Mexico/4108/2009 | 1.4E+07 TCID50/mL | + | + |
| B/Florida/04/2006 | 5.24E+05 TCID50/mL | + | + |
| Adenovirus1/Adenoid 71 | 5.67E+04 TCID50/mL | + | + |
| Adenovirus 2 | 2.51E+09 TCID50/mL | + | + |
| Adenovirus 3 | 1.10E+06 TCID50/mL | + | + |
| Adenovirus 4 | 9.54E+06TCID50/mL | + | + |
| Adenovirus 5 | 4.00E+06 TCID50/mL | + | + |
| Adenovirus 7 | 1.25E+07 TCID50/mL | + | + |
| Adenovirus 11 | 3.85E+06 TCID50/mL | + | + |
| Adenovirus 14 | 8.63E+04 TCID50/mL | + | + |
| Adenovirus 22 | N/A | + | + |
| Adenovirus 31 | 1.25E+05 TCID50/mL | + | + |
| Adenovirus 35 | N/A | + | + |
| Coronavirus NL63 | 1.41E+04 TCID50/mL | + | + |
| Coronavirus 229E | 1.70E+06TCID50/mL | + | + |
| Coronavirus OC43 | 1.67E+06 TCID50/mL | + | + |
| Coxsackievirus B4 | 2.43E+06 TCID50/mL | + | + |
| Coxsackievirus B5/10/2006 | 2.28E+06 TCID50/mL | + | + |
| Cytomegalovirus | 8.76E+05 TCID50/mL | + | + |
| Echovirus 7 | 5.38E+08 TCID50/mL | + | + |
| Echovirus 9 | 1.50E+06 TCID50/mL | + | + |
| Echovirus 6 | 1.05E+08 TCID50/mL | + | + |
| Echovirus 11 | 1.05E+05 TCID50/mL | + | + |
| Enterovirus 71 | 2.68E+03 TCID50/mL | + | + |
| Enterovirus 70 | 1.66E+05 TCID50/mL | + | + |
| Epstein Barr Virus | 5,000cp/mL | + | + |
| HSV Type 1 MacInytre | 1.95E+06 TCID50/mL | + | + |
| Human Rhinovirus | 1.26E+05 TCID50/mL | + | + |
| HSV Type 2 G strain | 3.67E+06 TCID50/mL | + | + |
| Measles virus | 1.95E+06 TCID50/mL | + | + |

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| Mumps virus | 2.75E+08 TCID50/mL | + | + |
| --- | --- | --- | --- |
| Parainfluenza Type 1 | 2.50E+05 TCID50/mL | + | + |
| Parainfluenza Type 2 | 2.20E+04 TCID50/mL | + | + |
| Parainfluenza Type 3 | 9.10E+05 TCID50/mL | + | + |
| Parainfluenza Type 4 | 1.36E+07 TCID50/mL | + | + |
| Varicella Zoster Virus | 7.5E+02TCID50/mL | + | + |
| Bordetella pertussis | 7.6E+08 CFU/mL | + | + |
| Bordetella bronchiseptica | 1.65E+07 CFU/mL | + | + |
| Chlamydophila pneumoniae | 1.0E+3 DNA copies/ul | + | + |
| Chlamydia trachomatis | 2.10E+05 TCID50/mL | + | + |
| Legionella pneumophila | 1.36E+07 CFU/mL | + | + |
| Mycobacterium intracellulare | 4.30E+06 CFP/mL | + | + |
| Mycobacterium tuberculosis | 1.10E+07 CFU/mL | + | + |
| Mycobacterium avium | 3.07E+05 CFU/mL | + | + |
| Mycoplasma pneumoniae | 4.64E+06 CCU/mL | + | + |
| Haemophilus influenzae | 3.60E+06 CFU/mL | + | + |
| Pseudomonas aeruginosa | 2.71E+06 CFU/mL | + | + |
| Proteus vulgaris | 5.20E+06 CFU/mL | + | + |
| Proteus mirabilis | 2.39E+06 CFU/mL | + | + |
| Neisseria gonorrhoeae | 6.90E+02 CFU/mL | + | + |
| Neisseria menigitidis | 3.60E+06 CFU/mL | + | + |
| Neisseria mucosa | 7.60E+06 CFU/mL | + | + |
| Klebsiella pneumoniae | 3.90E+06 CFU/mL | + | + |
| Escherichia coli | 3.30E+06 CFU/mL | + | + |
| Moraxella catarrhalis | 2.98E+03 CFU/mL | + | + |
| Corynebacterium diphtheriae | 2.39E+06 CFU/mL | + | + |
| Lactobacillus plantarum | 1.06E+06 CFU/mL | + | + |
| Streptococcus pneumoniae | 9.50E+05 CFU/mL | + | + |
| Streptococcus pyogenes | 2.43E+06 CFU/mL | + | + |
| Streptococcus salivarius | 1.49E+06 CFU/mL | + | + |
| Staphylococcus epidermidis | 7.50E+05 CFU/mL | + | + |
| Staphylococcus aureus | 7.80E+06 CFU/mL | + | + |
| Candida albicans | 1.79E+05 CFU/mL | + | + |

# Cross Reactivity:

To evaluate the potential cross reactivity of the test with other common respiratory pathogens, the organisms in the table above were tested. Samples were prepared by adding the potential interference organism into negative clinical matrix at $3\mathrm{x}$ LoD. The potential interference organisms were tested at one clinically relevant concentration, for viruses $10^{4} - 10^{6}$ TCID50/mL

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and for bacteria $10^{6} - 10^{8}$ CFU/mL (the same titers as in the Interference table above). The prepared samples were extracted and run on both the Cepheid SmartCycler II and ABI 7500 Fast Dx.

There was no cross-reactivity seen between any of the organisms tested, all tests were negative for both RSV or hMPV.

# Inclusivity:

Each virus was diluted into negative clinical matrix at 1x-3x LoD. Samples were extracted and tested in triplicate on either the ABI 7500 Fast Dx or Cepheid SmartCycler II. Virus was detected in all positive samples in all but one virus strain, hMPV B1 Peru3. Only 2/3 samples were positive during testing of hMPV B1 Peru 3. This result was probably due to a combination of small differences in LoD among different genetic sub-lineage and titer variability near the LoD. The study adequately shows that the assay can detect the two RSV subtypes and four hMPV genetic sub-lineages.

| RSV Inclusivity Panel | | | | |
| --- | --- | --- | --- | --- |
| Subtype | Strain | TCID50/mL | (ABI 7500 Fast Dx) | (Cepheid SmartCycler II) |
| A (NIBSC) | A-2 | N/A | Positive | Positive |
| A | A-2 | 5.67E+00 | Positive | Positive |
| B | Washington | 2.25E+00 | Positive | Positive |
| B | 9320 | 2.25E+00 | Positive | Positive |
| B | CH9318(1B) | 2.25E+00 | Positive | Positive |
| hMPV Inclusivity Panel | | | | |
| --- | --- | --- | --- | --- |
| Subtype | Strain | TCID50/mL | (ABI 7500 Fast Dx) | (Cepheid SmartCycler II) |
| A1 | IA3-2002 G Gene | 7.95E+01 | Positive | Positive |
| A1 | IA10-2003 | 7.95E+01 | Positive | Positive |
| A2 | IA14-2003 G Gene | 1.25E+1 | Positive | Positive |
| A2 | Clinical Isolate | 1.25E+1 | Positive | Positive |
| B1 | Peru2-2002 G Gene | 2.36E+01 | Positive | Positive |
| B1 | Peru3-2003 G Gene | 2.36E+01 | Positive | Positive |

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The Quidel Molecular RSV + hMPV assay detected 100% of the RSV and hMPV on both the ABI 7500 Fast Dx and Cepheid SmartCycler® II platforms.

f. Assay cut-off:

Threshold: The threshold for the ABI 7500 Fast Dx was established using the background fluorescence from cycle 3-15. The threshold is set to above the background fluorescence, below the plateau and linear regions of the amplification curve, within the exponential phase of the amplification curve. The threshold is designed to maximize sensitivity and specificity. The threshold settings for each analyte were determined individually using positive and negative clinical specimens.

The threshold for the Cepheid SmartCycler II was established using the background subtraction performed by the software algorithm and adjusting the threshold based on amplification curves obtained from positive and negative clinical samples.

Assay cut-off: The Ct cut-off was determined after testing clinical specimens and choosing a Ct value that was higher than the Ct value of the lowest positive concentration of analyte.

| Summary of Threshold Settings for the Quidel Molecular RSV + hMPV Assay by Instrument | | |
| --- | --- | --- |
| | ABI 7500 Fast Dx | SmartCycler |
| hMPV | 5.4e+04 | 10 |
| RSV | 8.0e+04 | 20 |
| MS2 | 2.4e+04 | 10 |
| Recommended Ct Cut-Off Values by Instrument | | | | |
| --- | --- | --- | --- | --- |
| | ABI 7500 Fast Dx | | SmartCycler | |
| | Latest Ct Value Observed* | Recommended Ct Cut-Off | Latest Ct Value Observed | Recommended Ct Cut-Off |
| hMPV | 5.4e+04 | 10 | 5.4e+04 | 10 |
| RSV | 8.0e+04 | 20 | 8.0e+04 | 20 |
| MS2 | 2.4e+04 | 10 | 2.4e+04 | 10 |

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15

| hMPV | 24.66 | 35 | 38.00 | 50 |
| --- | --- | --- | --- | --- |
| RSV | 30.08 | 35 | 49.70 | 50 |

*These values reflect subtraction of first the 10 cycles

## 2. Comparison studies:

### a. Method comparison with predicate device:

Method comparison was performed against the gold standard of culture for RSV testing and a FDA cleared device for hMPV. The testing description and data are listed in the Clinical studies section.

### b. Matrix comparison:

A transport media study was conducted to show efficacy of 5 different transport media (UTM, M4, M4-RT, M5 and M6). RSV and hMPV were spiked into the media at 3x LoD. Each condition was extracted in triplicate, and each extraction was tested twice. There was not statistical difference in Ct value among the media tested on either the ABI 7500 Fast Dx or the Cepheid SmartCycler II.

## 3. Clinical studies:

### a. Clinical Sensitivity and Specificity:

The clinical study for this device was performed during the 2012 respiratory virus season, January – March, 2012. All 1014 nasal swab and nasopharyngeal swap specimens were collected prospectively. One specimen per patient was collected at four US study sites. A total 414 samples were tested fresh and 600 were frozen and tested at a later date. Reference testing for RSV was DSFA or culture with DFA, performed immediately after collection. A FDA cleared hMPV molecular test was used as the hMPV comparator for the Quidel Molecular RSV + hMPV Assay.

The performance of the Quidel assay in detecting RSV and hMPV was acceptable. The Quidel assay detected an additional 21 and 28 positive RSV samples not detected by DSFA and cell culture with DFA respectively. This was confirmed by an FDA-cleared RT-PCR assay. The detection of hMPV was equivalent to a FDA cleared hMPV molecular test. Discrepant samples were not further tested for hMPV.

| Cepheid Smart Cycler II Combined Site - Respiratory syncytial virus | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | DSFA & Cell Culture w/DFA | | | | | 95% CI | |
| | | POS | NEG | Total | Sensitivity | 97.9% | 93.9% | 99.3% |
| QM RSV + hMPV | POS | 137 | 21* | 158 | Specificity | 97.6% | 96.3% | 98.4% |
| | NEG | 3 | 849 | 852 | | | | |
| | Total | 140 | 870 | 1010 | | | | |

* All originally discordant specimens were positive for RSV by an FDA-cleared RT-PCR assay.

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| Cepheid SmartCycler II Combined Site - Human metapneumovirus | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | FDA Cleared hMPV Molecular Test | | | | | 95% CI | |
| | | POS | NEG | Total | Positive percent agreement | 96.7% | 92.4% | 98.6% |
| QM RSV + hMPV | POS | 145 | 3 | 148 | Negative percent agreement | 99.6% | 98.9% | 99.9% |
| | NEG | 5 | 798 | 803 | | | | |
| | Total | 150 | 801 | 951 | | | | |
| ABI 7500 Fast Dx Combined Site - Respiratory syncytial virus | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | DSFA & Cell Culture w/DFA | | | | | 95% CI | |
| | | POS | NEG | Total | Sensitivity | 98.6% | 94.9% | 99.6% |
| QM RSV + hMPV | POS | 138 | 28* | 166 | Specificity | 96.8% | 95.4% | 97.8% |
| | NEG | 2 | 840 | 842 | | | | |
| | Total | 140 | 868 | 1008 | | | | |

* All originally discordant specimens were positive for RSV by an FDA-cleared RT-PCR assay.

| ABI 7500 Fast Dx Combined Site - Human metapneumovirus | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | FDA Cleared hMPV Molecular Test | | | | | 95% CI | |
| | | POS | NEG | Total | Positive percent agreement | 98.0% | 94.3% | 99.3% |
| QM RSV + hMPV | POS | 147 | 9 | 156 | Negative percent agreement | 98.9% | 97.9% | 99.4% |
| | NEG | 3 | 790 | 793 | | | | |
| | Total | 150 | 799 | 949 | | | | |

4. Clinical cut-off: N/A

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5. Expected values/Reference range:

The prevalence detected for RSV and hMPV during the 2012 respiratory virus season was approximately 16% and 15% respectively. Prevalence was higher for both viruses in children under 5 years of age. The table below shows the overall prevalence and the prevalence for all sites segregated by age group. The prevalence detected by this device is appropriate for expected values for RSV and hMPV during the winter months.

| Applied BioSystem 7500 Fast Dx | | | | | | Cepheid SmartCycler II | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Age Group (years) | RSV Positive | Prevalence | hMPV Positive | Prevalence | total tested | RSV Positive | Prevalence | hMPV Positive | Prevalence | total tested |
| <1 | 72 | 28.3% | 38 | 15.0% | 254 | 71 | 27.5% | 35 | 13.6% | 258 |
| 1 to 5 | 72 | 19.9% | 76 | 21.5% | 354 | 67 | 19.0% | 73 | 20.7% | 352 |
| 6 to 10 | 6 | 4.5% | 17 | 12.7% | 134 | 6 | 4.5% | 16 | 11.9% | 134 |
| 11 to 15 | 3 | 4.8% | 7 | 11.3% | 62 | 3 | 4.8% | 6 | 9.7% | 62 |
| 16 to 21 | 3 | 9.1% | 1 | 3.0% | 33 | 3 | 9.1% | 1 | 3.0% | 33 |
| >21 | 10 | 5.7% | 18 | 10.3% | 175 | 8 | 4.6% | 17 | 9.7% | 175 |
| Total | 166 | 16.4% | 157 | 15.5% | 1012* | 158 | 15.6% | 148 | 14.6% | 1014 |

N. Proposed Labeling:

The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.

O. Conclusion:

The submitted information in this premarket notification is complete and supports a substantial equivalence decision.